Project Summary Germ cells and somatic cells from an individual carry identical copies of DNA; yet, only germ cells have the potential to give rise to all the cell types of each subsequent generation. This suggests that epigenetic factors confer pluripotent and immortal potential to germ cells. Some of these epigenetic factors are exclusive to the germ cell cytoplasm, and in certain cases their presence is enough to reprogram somatic nuclei to restore pluripotency and immortal potential. A distinguishing feature of germ cell cytoplasm are germ granules. Germ granules are a phase-separated, heterogeneous mix of RNA and protein that have been observed in the germline of most animals. Because core germ-granule composition is conserved from nematodes to humans, the genetically tractable model C. elegans is used to study how germ granules function to regulate the pluripotent and immortal potential of germ cells. Germ granules extend the environment of the nuclear pore into the cytoplasm, where they serve as a safety next to ensure that nascent transcripts coming from the nucleus are licensed for germline expression. Depletion of germ granules in C. elegans causes sterility and germ-to-soma transformation. The long-term objectives of this research are to understand how germ granules regulate cellular pluripotency and their potential to be manipulated to induce pluripotency when it is needed (e.g. regeneration) or shut it off when it is not (e.g. cancer). A conserved core of germ-granule proteins act as multipotency factors, and are often repurposed in the soma during development, tissue regeneration, and tumorigenesis. This core consists of Vasa DEAD-box RNA helicases, small RNA binding Argonaute proteins, and LOTUS-Tudor domain proteins that interface and stimulate Vasa and Argonaute activity. The role of these multipotency factors during tumorigenesis, and whether they contribute to the phenomenon of cancer/testis (CT) antigen expression in various tumors has not been explored. Here, C. elegans is used to understand how these core proteins are expressed and distributed, what they interact with, and their role in fertility and developmental plasticity. The specific aims in this proposal look at 1) the role of germ granules in protein expression and turnover, primarily focusing on the novel interaction of GLH/Vasa with PCI scaffolding complexes, 2) how GLH promotes the differential translation of spermatogenic transcripts, and 3) how GLH and a new LOTUS-Tudor protein called LOTR-1 cooperates to maintain germline integrity. If successful, these findings will reveal novel ways to manipulate cellular pluripotency and expose the potential therapeutic targets that directly regulate the cytoplasm instead of nuclear gene expression networks.